Limits...
In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates.

Garrett DJ, Larson JE, Dunn D, Marrero L, Cohen JC - BMC Biotechnol. (2003)

Bottom Line: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates.Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts.Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ochsner Children's Research Institute, Ochsner Clinic Foundation, New Orleans, LA 70121, USA. djgarre@aol.com

ABSTRACT

Background: Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes.

Methods: Recombinant AAV2 carrying green fluorescent protein (GFP) was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor.

Results: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year.

Conclusions: Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

Show MeSH

Related in: MedlinePlus

GFP-specific sequences in rat lung at 12 months of age. DNA was extracted from rat lungs. Undigested DNA (10 μg) was electrophoresed on agarose gel for Southern blot analysis. DNA from rAAV2gfp infected 293 cells was used as a positive control. Lanes labeled AAV-GFP represents DNA from separate animals infected at 16 days gestation with rAAV2gfp. Negative Control labeled lanes are from age-matched animals not infected with an rAAV2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC239997&req=5

Figure 3: GFP-specific sequences in rat lung at 12 months of age. DNA was extracted from rat lungs. Undigested DNA (10 μg) was electrophoresed on agarose gel for Southern blot analysis. DNA from rAAV2gfp infected 293 cells was used as a positive control. Lanes labeled AAV-GFP represents DNA from separate animals infected at 16 days gestation with rAAV2gfp. Negative Control labeled lanes are from age-matched animals not infected with an rAAV2.

Mentions: Rats were followed up to one year of age, and DNA was extracted from both the lungs and intestines. Although the intestines are a primary target of in utero gene transfer from the amniotic fluid, no GFP DNA was detected by Southern blot in these tissues. This indicated that the dilution of transgene had decreased to levels below detection and /or that the original transfer efficiency of the intestine multipotential stem cells was poor. In contrast, GFP DNA was found in all the rats' lungs at 12 months of age as demonstrated in Fig. 3.


In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates.

Garrett DJ, Larson JE, Dunn D, Marrero L, Cohen JC - BMC Biotechnol. (2003)

GFP-specific sequences in rat lung at 12 months of age. DNA was extracted from rat lungs. Undigested DNA (10 μg) was electrophoresed on agarose gel for Southern blot analysis. DNA from rAAV2gfp infected 293 cells was used as a positive control. Lanes labeled AAV-GFP represents DNA from separate animals infected at 16 days gestation with rAAV2gfp. Negative Control labeled lanes are from age-matched animals not infected with an rAAV2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239997&req=5

Figure 3: GFP-specific sequences in rat lung at 12 months of age. DNA was extracted from rat lungs. Undigested DNA (10 μg) was electrophoresed on agarose gel for Southern blot analysis. DNA from rAAV2gfp infected 293 cells was used as a positive control. Lanes labeled AAV-GFP represents DNA from separate animals infected at 16 days gestation with rAAV2gfp. Negative Control labeled lanes are from age-matched animals not infected with an rAAV2.
Mentions: Rats were followed up to one year of age, and DNA was extracted from both the lungs and intestines. Although the intestines are a primary target of in utero gene transfer from the amniotic fluid, no GFP DNA was detected by Southern blot in these tissues. This indicated that the dilution of transgene had decreased to levels below detection and /or that the original transfer efficiency of the intestine multipotential stem cells was poor. In contrast, GFP DNA was found in all the rats' lungs at 12 months of age as demonstrated in Fig. 3.

Bottom Line: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates.Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts.Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ochsner Children's Research Institute, Ochsner Clinic Foundation, New Orleans, LA 70121, USA. djgarre@aol.com

ABSTRACT

Background: Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes.

Methods: Recombinant AAV2 carrying green fluorescent protein (GFP) was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor.

Results: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year.

Conclusions: Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

Show MeSH
Related in: MedlinePlus